CN104585027A - Seedling hardening method for ginger tissue culture seedlings - Google Patents

Abstract

The invention discloses a method for hardening ginger tissue-cultured seedlings, which comprises the following steps: (1) selecting healthy gingers free from diseases and insect pests as tissue culture materials, sterilizing ginger pieces and burying them in a sand bed to accelerate germination, sterilizing, and stripping 0.3 ～0.5mm shoot tips for inoculation; (2) The shoot tips are cultured through sterile seedling medium, induction medium, proliferation medium and rooting medium in sequence to form tissue culture bottle seedlings that can go out of the culture room; (3 ) When the tissue culture seedlings in the culture bottle have 4-5 white roots, remove the cover and harden the seedlings; 5 days later, move the culture bottle out of the culture room, and harden the seedlings under natural light and temperature of 24-26°C for 7 days. When the tissue-cultured seedlings have 5-6 leaves and the leaves are 5-7cm long, take out the tissue-cultured seedlings from the culture bottle and wash the medium, and transplant them into the matrix of peat: perlite: vermiculite = 2:1:1 , according to conventional fertilizer and water management until the finished seedlings grow into finished seedlings and come out of the nursery. The method is simple, easy to implement, low in cost and high in transplanting survival rate.

Description

A kind of seedling hardening method of ginger tissue culture seedling

technical field

The invention relates to a method for cultivating ginger, in particular to a method for hardening ginger tissue-cultured seedlings.

Background technique

Ginger (Zi ngi ber offici nale Rose) belongs to the Zingiberaceae Zingiberaceae perennial herb and is also an annual vegetable crop. Ginger is rich in nutrition and has a unique flavor, which can be used as an important condiment. Ginger is also in high demand due to its medicinal properties [1] and its popularity in the market. In recent years, with the increase of ginger export trade volume, the planting area has also expanded year by year. However, in production, ginger has been asexually propagated as underground tubers for a long time, and the reproduction coefficient is low [2-3]. Years of vegetative planting have caused infection by viruses and bacteria, and viruses [4-6] and Pseudomonas solanacearum are very easy to accumulate in the body through ginger, leading to the degradation of ginger varieties, decreased vitality, and stress resistance. It is easy to induce various diseases such as ginger blast and ginger mange, and the incidence rate in the field can be as high as 70% to 80%, resulting in poor quality and unstable content of medicinal ingredients, which poses a serious threat to ginger production. In recent years, great success has been achieved in ginger detoxification and virus propagation using tissue culture technology. In conventional cultivation, 4,500-6,000kg/hm2 of ginger is needed to plant germinated ginger in pieces, and the consumption of ginger is very large; therefore, it is bound to be a trend to use ginger bud tissue for tissue culture in the future. . However, there are still relatively few reports on the cultivation of ginger tissue culture seedlings, domestication and hardening methods.

Contents of the invention

The object of the present invention is to provide a simple and easy method for hardening ginger tissue cultured seedlings with low cost and high survival rate.

In order to achieve the above object, the present invention adopts the following technical solutions:

A kind of seedling hardening method of ginger tissue culture seedling, comprises the following steps successively:

(1) Treatment of seedling tissue:

Choose robust, pest-free ginger as tissue culture material, soak the ginger pieces in 50% carbendazim 500 times solution for half an hour, sterilize them, bury them in a clean sand bed, and germinate them at room temperature. When the size is 1-2 cm, rinse with distilled water to remove the sand, peel off the young leaves with a dissecting needle in a sterile room, take 3-5 mm ginger buds, sterilize with 70% alcohol for one minute, and then sterilize with 1%-10NaClO for 20 minutes. Rinse 3 times with distilled water, peel off the shoot tip with a dissecting mirror on the ultra-clean workbench, and peel off the shoot tip of 0.3-0.5 mm for inoculation;

(2) Cultivation of tissue culture seedlings:

Inoculate the 0.3-0.5mm shoot tip in step (1) into the sterile seedling medium, and cultivate it under the tissue culture conditions of 25°C, light intensity 1500Lx, and light 12h/d until the shoot tip grows to 1-2cm When the aseptic explants are formed; the sterile seedling medium is: MS+sucrose 40g/L+agar 7g/L, and the pH value is 6.0;

Inoculate the sterile explants into the induction medium, and culture them under the tissue culture conditions of 25°C, light intensity 1500Lx, and light 12h/d for 22-25 days to form the first generation of adventitious buds and grow to 1.0-3.0cm; The induction medium described is: MS+zeatin 2mg/L+6_BA 1.5mg/L+NAA 0.15mg/L+IAA 0.1mg/L+sucrose 40g/L+agar 7g/L, pH value is 6.0

Inoculate the above-mentioned first-generation adventitious sprouts into the proliferation medium, and culture them under the tissue culture conditions of 25°C, light intensity 1500Lx, and light 12h/d for 10-15 days until the second-generation adventitious sprouts are formed and grow to 3.0-6.0cm ; Described proliferation medium is: MS+zeatin 1mg/L+6-BA 2mg/L+NAA 0.25mg/L+IAA 0.3mg/L+sucrose 40g/L+agar 7g/L, pH value is 6.0;

Inoculate the above-mentioned subcultured adventitious sprouts into the rooting medium, and culture them for 12-15 days under the tissue culture conditions of 25°C, light intensity 1500Lx, and light 12h/d. The seedlings grow to 4-8cm and have at least 3 long When the root is ≥ 2cm, it will be a tissue culture bottle seedling that can go out of the culture room; the rooting medium is: MS+ gibberellin 1mg/L+KT 2mg/L+NAA 0.1mg/L+IAA 0.05mg/L+ Sucrose 40g/L+agar 7g/L, pH 6.0;

(3) Domestication of tissue culture seedlings:

When the tissue culture seedlings in the culture bottle have 4-5 white roots, remove the cover to harden the seedlings, and add a small amount of distilled water to the culture bottle; after 5 days, move the culture bottle out of the culture room, and place it under natural light and at a temperature of 24-26°C. The seedlings were hardened for 7 days under the conditions, and when 5-6 leaves appeared in the tissue-cultured seedlings, and when the leaves were 5-7cm long, the tissue-cultured seedlings were taken out from the culture bottle and the culture medium was cleaned, and transplanted into peat: perlite: vermiculite= In the substrate of 2:1:1, manage with conventional fertilizer and water until the finished seedlings grow into finished seedlings and go out of the nursery.

The present invention adopts a new formula culture medium different from the traditional culture medium in the stage of tissue culture; multiple growth hormones interact synergistically, allowing the ginger tissue to germinate and grow roots, and after transplanting, its survival rate is higher than that of the previous high.

MS medium:

6_BA: namely 6-benzylaminoadenine, is a kind of cytokinin, which mainly causes cell division, induces the formation of buds and promotes the growth of buds.

NAA: Naphthaleneacetic acid (1-Naphthaleneacetic acid), referred to as NAA, is an organic compound (chemical formula: C10H7CH2CO2H), a colorless solid that is easily soluble in organic solvents. Its structure is that the No. 1 position of naphthalene is substituted with carboxymethyl. It is a plant hormone auxin commonly used in commercial rooting powders or lotions when plants are propagated by cuttings. It can also be used in plant tissue culture.

IAA: Indole-3-acetic acid, is an endogenous auxin ubiquitous in plants and belongs to indole compounds. At the cellular level, auxin can stimulate cambium cell division; stimulate branch cell elongation and inhibit root cell growth; promote xylem and phloem cell differentiation, promote rooting of cuttings, and regulate callus morphogenesis.

KT: The chemical name is 6-glycosylaminopurine (or N6-furylmethyladenine), and the molecular formula is C10H9N5O; it is a non-natural cytokinin that can promote cell differentiation, division, and growth; induce callus growth Budding; relieve apical dominance; promote seed germination, break dormancy of lateral buds.

Zeatin: a natural cytokinin, it is the first natural cytokinin extracted and crystallized from the grains of sweet corn; it promotes callus germination; it can be synthesized artificially.

Gibberellins are a class of plant hormones that exist widely. Its chemical structure belongs to diterpenoid acids, derived from a tetracyclic skeleton. It is a new generation of new high-tech regulator product successfully developed in combination with international technology. Gibberellin is an improved drug of 920. The English name: gibberellin, referred to as GA4+7, its efficacy on crops is 100%. The effect is long-lasting, more efficient, more stable, and safer. Spraying at the seedling stage is the best, which can make the root system develop and prevent diseases. It can significantly promote the growth of plant stems and leaves. If spraying during the growth period, it can also make Balanced nutrition is helpful for crop growth. Spraying during the flowering period can preserve flowers and fruits, and can also make the fruit swell and have a more beautiful effect. Spraying during the full-blooming period of cotton can effectively reduce the shedding of buds and bells, increase the rate of bolling, and can effectively remove crop disease

Advantages of the present invention:

1. The seedling hardening method of the present invention is simple and easy to implement, has low cost and high transplanting survival rate.

2. Choose ginger stem tips as tissue culture materials. Ginger tissue culture seedlings have the characteristics of fast growth, vigorous growth, disease resistance, strong stress resistance, bright yellow color, uniform and neat ginger, strong spicy taste, good quality, and high yield.

3. In the stage of tissue culture, a new formula medium with synergistic effects of various hormones is used, and different hormones are selected according to different stages, which has a better effect than the traditional formula medium. For example, adding zeatin to the induction medium can promote more germination of ginger tissues; adding gibberellin to the rooting medium can promote the most rooting of tissue culture seedlings, which helps to improve the survival rate of tissue culture seedlings transplanted.

Detailed ways

Below in conjunction with embodiment the present invention is further described.

Example 1:

A kind of seedling hardening method of ginger tissue culture seedling, comprises the following steps successively:

(1) Treatment of seedling tissue:

Choose robust, pest-free ginger as tissue culture material, soak the ginger pieces in 50% carbendazim 500 times solution for half an hour, sterilize them, bury them in a clean sand bed, and germinate them at room temperature. When it is about 1 cm long, use distilled water to wash away the sand, peel off the young leaves with a dissecting needle in a sterile room, take about 3 mm ginger buds, sterilize with 70% alcohol for one minute, then sterilize with 1% NaClO for 20 minutes, rinse with distilled water for 3 Second, peel off the shoot tip with a dissecting mirror on the ultra-clean workbench, and strip off the shoot tip of about 0.3mm for inoculation;

(2) Cultivation of tissue culture seedlings:

Inoculate the shoot tip of about 0.3mm in step (1) into the sterile seedling medium, and cultivate it under the tissue culture conditions of 25°C, light intensity 1500Lx, and light 12h/d until the shoot tip grows to about 1cm. Explant sterile seedlings; the sterile seedling culture medium is: MS+sucrose 40g/L+agar 7g/L, pH value is 6.0;

Inoculate the explanted sterile seedlings into the induction medium, and culture them for 22 days under the tissue culture conditions of 25°C, light intensity 1500Lx, and light 12h/d until the first generation of adventitious buds are formed and grow to about 1cm; the induction culture The base is: MS+zeatin 2mg/L+6_BA 1.5mg/L+NAA 0.15mg/L+IAA 0.1mg/L+sucrose 40g/L+agar 7g/L, pH value is 6.0

Inoculate the above-mentioned first-generation adventitious sprouts into the proliferation medium, and culture them for about 10 days under the tissue culture conditions of 25°C, light intensity 1500Lx, and light 12h/d until the second-generation adventitious sprouts are formed and grow to about 3cm; The proliferation medium is: MS+zeatin 1mg/L+6_BA 2mg/L+NAA 0.25mg/L+IAA 0.3mg/L+sucrose 40g/L+agar 7g/L, pH value is 6.0;

Inoculate the above-mentioned subcultured adventitious sprouts into the rooting medium, and culture them for 12 days under the tissue culture conditions of 25°C, light intensity 1500Lx, and light 12h/d. When rooting, it will be a tissue culture bottle seedling that can go out of the culture room; the rooting medium is: MS+ gibberellin 1mg/L+KT 2mg/L+NAA 0.1mg/L+IAA 0.05mg/L+ sucrose 40g/L L+agar 7g/L, pH 6.0;

(3) Domestication of tissue culture seedlings:

When the tissue cultured seedlings in the culture bottle have 4 white roots, remove the cover to harden the seedlings, and add a small amount of distilled water to the culture bottle; after 5 days, move the culture bottle out of the culture room, and harden the seedlings under natural light and a temperature of 24°C After 7 days, when the tissue cultured seedlings had 5 leaves and the leaf length was about 5 cm, the tissue cultured seedlings were taken out from the culture bottle and the culture medium was cleaned, and transplanted into the matrix of peat: perlite: vermiculite = 2:1:1 During the process, the conventional fertilizer and water management is used until the finished seedlings grow into finished seedlings and come out of the nursery.

Example 2:

A kind of seedling hardening method of ginger tissue culture seedling, comprises the following steps successively:

(1) Treatment of seedling tissue:

Choose robust, pest-free ginger as tissue culture material, soak the ginger pieces in 50% carbendazim 500 times solution for half an hour, sterilize them, bury them in a clean sand bed, and germinate them at room temperature. When it is about 2cm, use distilled water to wash away the sand, peel off the young leaves with a dissecting needle in a sterile room, take 3-5mm ginger buds, sterilize with 70% alcohol for one minute, then sterilize with 10% NaClO for 20 minutes, and rinse with distilled water 3 times, peel off the shoot tip with a dissecting mirror on the ultra-clean workbench, and peel off the shoot tip of about 0.5mm for inoculation;

(2) Cultivation of tissue culture seedlings:

Inoculate the shoot tip of about 0.5mm in step (1) into the sterile seedling medium, and cultivate it under the tissue culture conditions of 25°C, light intensity 1500Lx, and light 12h/d until the shoot tip grows to about 2cm. Explant sterile seedlings; the sterile seedling culture medium is: MS+sucrose 40g/L+agar 7g/L, pH value is 6.0;

Inoculate the explant sterile seedlings into the induction medium, and cultivate them for 25 days under the tissue culture conditions of 25°C, light intensity 1500Lx, and light 12h/d until the first generation of adventitious buds are formed and grow to about 3.0cm; the induction Medium: MS+zeatin 2mg/L+6_BA 1.5mg/L+NAA 0.15mg/L+IAA 0.1mg/L+sucrose 40g/L+agar 7g/L, pH 6.0

Inoculate the above-mentioned first-generation adventitious sprouts into the proliferation medium, and culture them for 15 days under the tissue culture conditions of 25°C, light intensity 1500Lx, and light 12h/d until the adventitious sprouts of the second generation are formed and grow to about 6.0cm; The proliferation medium is: MS+zeatin 1mg/L+6_BA 2mg/L+NAA 0.25mg/L+IAA 0.3mg/L+sucrose 40g/L+agar 7g/L, pH value is 6.0;

Inoculate the above-mentioned subcultured adventitious sprouts into the rooting medium, and culture them for 15 days under the tissue culture conditions of 25°C, light intensity 1500Lx, and light 12h/d. When rooting, it will be a tissue culture bottle seedling that can go out of the culture room; the rooting medium is: MS+ gibberellin 1mg/L+KT 2mg/L+NAA 0.1mg/L+IAA 0.05mg/L+ sucrose 40g/L L+agar 7g/L, pH 6.0;

(3) Domestication of tissue culture seedlings:

When the tissue cultured seedlings in the culture bottle have 5 white roots, remove the cover to harden the seedlings, and add a small amount of distilled water to the culture bottle; after 5 days, remove the culture bottle from the culture room, and harden the seedlings under natural light and a temperature of 26°C On the 7th day, when the tissue culture seedlings appeared 6 leaves, and the leaf length was about 7cm, the tissue culture seedlings were taken out from the culture bottle and the culture medium was cleaned, and transplanted into the matrix of peat: perlite: vermiculite=2:1:1 During the process, the conventional fertilizer and water management is used until the finished seedlings grow into finished seedlings and come out of the nursery.

Example 3:

A kind of seedling hardening method of ginger tissue culture seedling, comprises the following steps successively:

(1) Treatment of seedling tissue:

Choose robust, pest-free ginger as tissue culture material, soak the ginger pieces in 50% carbendazim 500 times solution for half an hour, sterilize them, bury them in a clean sand bed, and germinate them at room temperature. When the size is 1.5cm, use distilled water to wash away the sand, peel off the young leaves with a dissecting needle in a sterile room, take 4mm ginger buds, sterilize with 70% alcohol for one minute, then sterilize with 5% NaClO for 20 minutes, rinse with distilled water 3 times , peel off the shoot tip with a dissecting mirror on the ultra-clean workbench, and peel off the shoot tip of 0.4mm for inoculation;

(2) Cultivation of tissue culture seedlings:

Inoculate the 0.4mm shoot tip in step (1) into the sterile seedling medium, and cultivate it under the tissue culture conditions of 25°C, light intensity 1500Lx, and light 12h/d until the shoot tip grows to 1.5cm. Implant sterile seedlings; the sterile seedling culture medium is: MS+sucrose 40g/L+agar 7g/L, pH value is 6.0;

Inoculate the explant sterile seedlings into the induction medium, and cultivate them for 24 days under the tissue culture conditions of 25°C, light intensity 1500Lx, and light 12h/d until the first generation of adventitious buds are formed and grow to 2cm; the induction medium For: MS+zeatin 2mg/L+6_BA 1.5mg/L+NAA 0.15mg/L+IAA 0.1mg/L+sucrose 40g/L+agar 7g/L, pH value is 6.0

Inoculate the above-mentioned first-generation adventitious sprouts into the proliferation medium, and culture them for 13 days under the tissue culture conditions of 25°C, light intensity 1500Lx, and light 12h/d until the adventitious sprouts of the second generation are formed and grow to 5cm; the proliferation The medium is: MS+zeatin 1mg/L+6_BA 2mg/L+NAA 0.25mg/L+IAA 0.3mg/L+sucrose 40g/L+agar 7g/L, the pH value is 6.0;

Inoculate the above-mentioned subcultured adventitious sprouts into the rooting medium, and culture them for 13 days under the tissue culture conditions of 25°C, light intensity 1500Lx, and light 12h/d. The seedlings grow to 6cm and have at least 3 roots ≥ 2cm in length At the same time, it will be a tissue culture bottle seedling that can go out of the culture room; the rooting medium is: MS+ gibberellin 1mg/L+KT 2mg/L+NAA 0.1mg/L+IAA 0.05mg/L+ sucrose 40g/L+ Agar 7g/L, pH 6.0;

(3) Domestication of tissue culture seedlings:

When the tissue cultured seedlings in the culture bottle have 5 white roots, remove the cover to harden the seedlings, and add a small amount of distilled water to the culture bottle; after 5 days, remove the culture bottle from the culture room, and harden the seedlings under natural light and a temperature of 25°C After 7 days, when 6 leaves appeared in the tissue cultured seedlings and the leaves were 6 cm long, the tissue cultured seedlings were taken out from the culture bottle and the culture medium was cleaned, and transplanted into the substrate of peat: perlite: vermiculite = 2:1:1 , according to conventional fertilizer and water management until the finished seedlings grow into finished seedlings and come out of the nursery.

Claims (1)

1. a hardening off method for ginger plantlet in vitro, is characterized in that, comprises the following steps successively:

(1) process of seedling tissue:

Select healthy and strong, be tissue cultures material without the ginger of damage by disease and insect, by ginger block with after the 500 times of immersion bubble half an hour sterilizations of 50% carbendazim, be embedded in clean husky bed, carry out vernalization at ambient temperature, as the long 1 ~ 2cm of bud, distilled water flushing is used to remove sandy soil, spire is peelled off at aseptic indoor dissecting needle, get 3 ~ 5mm ginger bud, with 70% alcohol sterilizing one minute, then use 1% ~ 10NaClO sterilizing 20 minutes, distilled water flushing 3 times, superclean bench carries out stem apex stripping with anatomical lens, and the stem apex stripping 0.3 ~ 0.5mm is inoculated;

(2) cultivation of plantlet in vitro:

The stem apex of 0.3 ~ 0.5mm in step (1) is inoculated in Aseptic seedling culture base, 25 DEG C, light intensity 1500Lx, illumination 12h/d condition of tissue culture under cultivate, until become explant aseptic seedling when stem apex grows to 1 ~ 2cm; Described Aseptic seedling culture base is: MS+ sucrose 40g/L+ agar 7g/L, and pH value is 6.0;

Explant aseptic seedling is inoculated in inducing culture, 25 DEG C, light intensity 1500Lx, illumination 12h/d condition of tissue culture under cultivate and just grow to 1.0 ~ 3.0cm for indefinite bud to be formed in 22 ~ 25 days; Described inducing culture is: MS+ zeatin 2mg/L+6_BA 1.5mg/L+NAA 0.15mg/L+IAA 0.1mg/L+ sucrose 40g/L+ agar 7g/L, pH value is 6.0

Just be inoculated in proliferated culture medium for adventitious shoot by above-mentioned, 25 DEG C, light intensity 1500Lx, illumination 12h/d condition of tissue culture under cultivate 10 ~ 15 days to forming subculture adventitious shoot and growing to 3.0 ~ 6.0cm; Described proliferated culture medium is: MS+ zeatin 1mg/L+6_BA 2mg/L+NAA 0.25mg/L+IAA 0.3mg/L+ sucrose 40g/L+ agar 7g/L, and pH value is 6.0;

Above-mentioned subculture adventitious shoot is inoculated in root media, 25 DEG C, light intensity 1500Lx, illumination 12h/d condition of tissue culture under cultivate 12 ~ 15 days, when seedling grows to 4 ~ 8cm and at least has the root of 3 long >=2cm, can go out the tissue-culture container seedling of culturing room; Described root media is: MS+ gibberellin 1mg/L+KT 2mg/L+NAA 0.1mg/L+IAA 0.05mg/L+ sucrose 40g/L+ agar 7g/L, and pH value is 6.0;

(3) domestication of plantlet in vitro:

When plantlet in vitro in blake bottle is grown and had 4 ~ 5 white roots, take off lid hardening, and in blake bottle, add a small amount of distilled water; After 5 days, blake bottle is shifted out culturing room, at the condition lower refining seedling 7 days of natural lighting and temperature 24 ~ 26 DEG C, treat that 5 ~ 6 leaves appear in plantlet in vitro, during leaf length 5 ~ 7cm, in blake bottle, take out plantlet in vitro and clean medium, be transplanted into the peat composed of rotten mosses: perlite: in the matrix of vermiculite=2:1:1, rich water quality management is to growing up to finished product seedling routinely, goes out garden.